Thus, the present invention is directed to the production of proteins or enzymes of bacterial origin in filamentous fungi, for example in Aspergillus and Trichoderma, by using a fusion to a secretable fungal protein or one or more functional domains of said protein to obtain improved secretion of said bacterial protein or enzyme. Preferably, the proteins originate from actinomycetes. The compositions of the invention are useful for e.g. modifying plant biomass properties, especially to reduce the lignin content in enzyme aided bleaching. The invention is also directed to the area of thermostable xylanases that are active at high temperatures and to a method for bleaching with the aid of the enzyme compositions of the invention.
The aim of kraft pulp bleaching is to remove the residual lignin that is left in pulp after kraft cooking. Traditionally, this has been done by using chlorine-containing chemicals. Because of environmental concerns and consumer demands, alternative bleaching technologies have been desired.
The first biotechnical approach to this problem was to attack the lignin directly with lignin degrading enzymes. However, the chemistry of enzymatic lignin degradation seems to be very complicated and difficult to control.
Lignin can be degraded, if the whole microorganism that produces ligninolytic enzymes is used. However, treatment times are relatively long. For example, treatment times may take days, and the microorganisms need supplemental nutrients to work. It can also be difficult to control the growth of other, undesired, microbes. The use of lignin degradation by isolated ligninolytic enzymes or by microorganisms is the subject of much research. (see, for example, Farrell, R. L. et al., Lignocellulosics 305-315 (1992); Jurasek, L., Lignocellulosics 317-325 (1992)).
In addition to cellulose and lignin, wood pulp contains hemicellulose. Another approach to reduce the lignin content of pulp is to attack hemicellulose—the third main component of wood. The hemicellulose in native hardwood is mainly xylan, while in softwood the hemicellulose is mainly glucomannans and some xylan. During kraft cooking, part of the xylan is dissolved into the cooking liquor. Towards the end of the cooking period when the alkali concentration decreases, part of the dissolved and modified xylan reprecipitates back onto the cellulose fibre.
In 1986, it was noticed that xylanase pretreatment of unbleached kraft pulp results in a lessened need for chemicals in the bleaching process (Viikari, L. et al., Proceedings of the 3rd Int. Conf. on Biotechnology in the Pulp Paper Ind., Stockholm (1986), pp. 67-69). Xylanase pretreatment of kraft pulp partially hydrolyses the xylan in kraft pulp. The mechanism of how hydrolysis of xylan results in better lignin removal is not fully understood. One frequently suggested possibility is that the pulp structure becomes more porous and this enables more efficient removal of lignin fragments in the subsequent bleaching and extraction stages. Also hydrolysis of the xylan located in the inner parts of the fibre and possibly linked to lignin may have a role. Later, in several laboratories, the xylanase pretreatment was reported to be useful in conjunction with bleaching sequences consisting of Cl2, ClO2, H2O2, O2 and O3. See reviews in Viikari, L. et al., FEMS Microbiol. Rev. 13: 335-350 (1994); Viikari, L. et al., in: Saddler, J. N., ed., Bioconversion of Forest and Agricultural Plant Residues, C-A-B International (1993), pp. 131-182; Grant, R., Pulp and Paper Int. (Sept. 1993), pp. 56-57; Senior & Hamilton, J. Pulp & Paper:111-114 (Sept. 1992); Bajpai & Bajpai, Process Biochem. 27:319-325 (1992); Onysko, A., Biotech. Adv. 11: 179-198 (1993); and Viikari, L. et al., J. Paper and Timber 73:384-389 (1991).
As a direct result of the better bleachability of the pulp after such a xylanase treatment, there is a reduction of the subsequent consumption of bleaching chemicals, which when chlorine containing chemicals are used, leads to a reduced formation of environmentally undesired organo-chlorine compounds. Also as a direct result of the better bleachability of pulp after a xylanase treatment, it is possible to produce a product with a higher final brightness where such brightness would otherwise be hard to achieve (such as totally chlorine free (TCF) bleaching using peroxide). Because of the substrate specificity of the xylanase enzyme, cellulose fibers are not harmed and the strength properties of the product are well within acceptable limits.
A xylanase that is active at an alkaline pH would decrease the need to acidify the pulp prior to xylanase treatment. In addition, the temperatures of many modern kraft cooking and bleaching processes are relatively high, well above the 50° C. that is suitable for many of the commercial bleaching enzymes. Accordingly, a need exists for thermostable xylanase preparations that are stable at alkaline pH values for use in wood pulp bleaching processes.
It is known that actinomycetes, e.g. (Microtetraspora flexuosa ATCC35864 and Thermomonospora fusca KW3, produce thermostable and alkaline stable xylanases (U.S. Pat. No. 5,437,992 and EP 473 545. The cloning of xylanases has been reported from several bacteria (e.g. Ghangas, G.S. et al., J. Bacteriol. 171:2963-2969 (1989); Lin, L. -L., Thomson, J. A., Mol. Gen. Genet. 228:55-61 (1991); Shareck, F. et al., Gene 107:75-82 (1991); Scheirlinck, T. et al., Appl Microbiol Biotechnol. 33:534-541 (1990); Whitehead, T. R., Lee, D. A., Curr. Microbiol. 23:15-19 (1991)); and also from Actinomadura sp. FC7 (Ethier, J. -F. et al., in: Industrial Microorganisms: Basic and Applied Molecular Genetics, R. Baltz et al., Eds, (Proc. 5th ASM Conf. Gen. Mol. Biol. Indust. Microorg., Oct. 11-15, 1992, Bloomington, Ind., poster C25)). It has been proposed by some researchers that the former genus Actinomadura should be divided into two genera, Actinomadura and Microtetraspora, the latter including, e.g. the former A. flexuosa (Kroppenstedt et al., System. Appl. Microbiol. 13: 148-160 (1990).
The use of hemicellulose hydrolyzing enzymes in different bleaching sequences is discussed in WO 89/08738, EP 383 999, WO 91/02791, EP 395 792, EP 386 888, EP 473 545, EP 489 104 and WO 91/05908, WO 95/34662, WO 95/18219, WO 95/27779, WO 95/34662, WO 95/18219, WO 92/04664 and WO 92/03540. The use of hemicellulolytic enzymes for improved water removal from mechanical pulp is discussed in EP 262 040, EP 334 739 and EP 351 655, DE 4,000,558, WO 92/04664, WO 92/03540, WO 94/21785 and EP 513 140. When the hydrolysis of biomass to liquid fuels or chemicals is considered, the conversion of both cellulose and hemicellulose is essential to obtain a high yield (Viikari et al., “Hemicellulases for Industrial Applications,” In: Bioconversion of Forest and Agricultural Wastes, Saddler, J., ed., CAB International, USA (1993)). Also, in the feed industry, there is a need to use a suitable combination of enzyme activities to degrade the high β-glucan and hemicellulose containing substrate.
The efficient and cost-effective production of thermostable xylanases is a problem, because thermostable xylanases originate mainly from relatively unstudied bacteria, which often produce only minimal or very small amounts of xylanase. Further, there is little or no experience of cultivating these microbes in a fermentor or no fermentation processes available. Furthermore, these microbes may be unsuitable for industrial scale production. On the other hand, filamentous fungi like Aspergillus and Trichoderma are known to produce large quantities of proteins, on an industrial scale. In particular, these fungi have been shown to be suitable for production of homologous or heterologous proteins of fungal origin.
There are very few reports related to the production of proteins or enzymes of bacterial origin in filamentous fungi: the production of endoglucanase from Cellulomonas fimi (Gwynne et al., Bio/Technology 5: 713-719 (1987); and β-glucuronidase from E. coli (Punt et al., J. Biotechnol. 17: 19-34 (1991) have been reported in A. nidulans. Of these enzymes, endoglucanase was secreted into the culture medium by Aspergillus nidulans in the range of 10-15 mg protein per liter. β-glucuronidase was only detectable intracellularly.
Many of the studies on heterologous gene expression have concerned mammalian genes (van den Hondel et al., Heterologous gene expression in filamentous fungi, Ed. Bennett and Lasure. More Gene Manipulations in Fungi Academic Press, San Diego, U.S.A., pp. 396-428 (1991). So far, the initial yields of eucaryotic enzymes in filamentous fungi have been in a range of tens of mg per liter in shake flask cultivations. In the International patent publication WO 90/15860 secretion of chymosin by A. niger var. awamori was described using a fusion to the homologous glucoamylase gene. Nyyssönen et al., Bio/Technology 11: 591-595 (1993) describes the production of antibody fragments in Trichoderma reesei. The best yield of antibody fragments when produced as a fusion to the cellobiohydrolase 1 gene of T. reesei was in the range of 40 mg per liter in a shake flask cultivation.
So far the inventors of the present application are not aware of any reports of the production of proteins of bacterial origin in Trichoderma. 